I. Field of Invention
The present invention relates generally to the fields of immunology, microbiology, bacteriology, and molecular biology. More specifically, the present invention relates to compositions and methods for Ehrlichia diagnostics and therapeutics.
II. Background
The term ehrlichiosis has been broadly applied to a variety of diseases of humans and animals caused by pathogens classified in the genus Ehrlichia. Ehrlichia chaffeensis causes human monocytic ehrlichiosis (HME). Ehrlichiosis is transmitted by the bite of infected ticks, including the lone star tick. HME was first reported in 1987. The clinical symptoms of HME include fever, headache, malaise, myalgia, rash, lymphadenopathy, and nausea (Rikihisa, 1999). HME can be fatal to the immune compromised and elderly. White-tailed deer are presumed to be the reservoir hosts of E. chaffeensis (Dawson et al., 1994; Lockhart et al., 1997).
Illness due to ehrlichiosis can be so mild that no medical care is sought or the illness can be severe and sometimes fatal. Symptoms are generally non-specific and other diagnoses may be considered. Because the laboratory tests that detect ehrlichiosis are often not positive in the first week of illness, physicians base early patient treatment decisions on the signs and symptoms, as well as the patient's history. The physician also looks at specific blood tests to help determine the likelihood of ehrlichiosis. Clues such as a low platelet count (thrombocytopenia), low serum sodium levels (hyponatremia), abnormal white blood cell counts (elevated or decreased), or elevated liver enzyme levels are often helpful predictors.
Serologic assays are the most frequently used methods for confirming cases of ehrlichiosis. The indirect immunofluorescence assay (IFA) is generally considered the reference standard in ehrlichiosis serology. Other assays include ELISA, latex agglutination, and dot immunoassays. Serologic tests can be used to detect either IgG or IgM antibodies. Blood samples taken early (acute) and late (convalescent) in the disease are the preferred specimens for evaluation. Most patients demonstrate increased IgM titers by the end of the first week of illness, but IgM assays may be falsely elevated due to other bacterial infections. IgG antibodies are considered more accurate for the ehrlichiosis, but detectable levels of IgG antibody generally do not appear until 7-10 days after the onset of illness. It is important to consider the amount of time it takes for antibodies to appear when ordering laboratory tests, especially because most patients visit their physician relatively early in the course of the illness, before diagnostic antibody levels may be present. The value of testing two sequential serum or plasma samples together to show a rising antibody level is important in confirming acute infection with ehrlichiosis. Because antibody titers may persist in some individuals for years after the original exposure, only demonstration of recent changes in titers between paired specimens can be considered reliable confirmation of an acute infection.
The most rapid and specific diagnostic assays for ehrlichiosis rely on molecular methods like PCR that can detect DNA present in a whole blood or tissue sample. PCR on whole blood specimens taken early during illness have been shown to be a very effective tool to diagnose ehrlichiosis. Immunostaining procedures can also be performed on formalin-fixed tissue samples. Ideally, whole blood or skin biopsy specimens used for diagnosis should be taken before or within the first 48 hours after doxycycline treatment is started; after antibiotic therapy has been started, it becomes more difficult to detect the organisms by these methods.
Canine ehrlichiosis is a disease of dogs and wild canids (e.g., wolves) and is found worldwide. Canine ehrlichiosis is also known by other names such as “tracker dog disease”, “tropical canine pancytopenia”, “canine hemorrhagic fever”, and “canine typhus”. Canine monocytic ehrlichiosis (CME) is an important tick-borne disease of dogs worldwide that is caused primarily by the obligatory intracellular organism Ehrlichia canis (Neer et al., 2002). E. chaffeensis can also infect dogs and several wild animals (Dawson et al., 1996) (Table 1). E. canis causes canine monocytic ehrlichiosis and was first recognized in Algeria in 1935 (Buhles et al., 1974). Wild and domestic dogs with chronic infection serve as reservoir hosts. During the acute phase of infection, the clinical signs include fever, anorexia, and lymphadenopathy, and, in the chronic phase of infection, the dogs may show emaciation, hemorrhage, and peripheral edema (Buhles et al., 1974).
Two blood tests that detect the dog's antibodies to Ehrlichia are available. One is called the indirect immunofluorescent antibody (IFA) test, and the other is ELISA test. A veterinarian cannot rely solely on these tests to make a diagnosis. The antibodies may not be detected in the early phase of the disease, since it takes some time for the body to make them. Also, if a dog is extremely ill, it may not be able to produce enough antibodies to be accurately detected. A positive test demonstrates that the dog has been exposed to Ehrlichia, but not that it necessarily is currently infected. In the acute stage of the disease, the antibody level will rise significantly. Often two tests will be done 2 weeks apart and the results compared. Dogs with an active infection will show a significant rise in the amount of antibody present. The antibodies can last for one or more years after the infection, but they do not make the dog immune to ehrlichiosis—the dog can be reinfected.
Techniques using PCR test for the presence of the organism itself, not antibodies to it. Unfortunately, it does not distinguish between live and dead organisms. For this reason, it is generally recommended to perform the PCR along with one of the antibody tests to make a diagnosis. There is no vaccine for ehrlichiosis currently. Thus, new methods and compositions are needed to diagnose and treat ehrlichiosis.